Isolation And Disease Resistance Analysis Of A Mitogen-activated Protein Kinase Gene GhMPK4 From Cotton

As an important economic crop in our country,cotton is often attacked by various pathogens,which affects the yield and quality of cotton.Fusarium wilt was caused by Fusarium oxysporum,which is the main disease affecting cotton production.In order to resist the invasion of diseases,plants have formed a series of response mechanisms during long-term evolution.The mitogen-activated protein kinase(MAPK)cascade pathway is ubiquitous in eukaryotes and is a highly conserved signal transduction pathway that plays an important role in pathogen-induced resistance responses.So far,there have been few reports on the studies on the resistance of MAPK to Fusarium wilt.Therefore,using molecular biology methods to research the regulation of MAPK cascade pathway to Fusarium wilt,which is of great theoretical and practical significance for improving the resistance of Fusarium wilt.In this study,we isolated a MAPK gene GhMPK4 from upland cotton.Through analysis of its expression characteristics and biological function,it was initially found that GhMPK4 plays an important role in the resistance to Fusarium wilt in cotton.The main results as followed:(1)The coding region sequence of GhMPK4 was obtained by PCR methods and the length was 1137 bp and encoded a 378 amino acids polypeptide.The predicted molecular weight of GhMPK4 was 43.37 kDa and the isoelectric point was 6.6.According to the amino acid sequence alignment and phylogenetic tree analysis,we found that GhMPK4 belonged to group B MAPK.(2)Subcellular localization experiments revealed that GhMPK4 is localized in the cytoplasm and nucleus,which indicated that GhMPK4 may perform its functions in the cytoplasm and nucleus.(3)The expression of Gh MPK4 was inhibited by F.oxysporum and plant hormones.The expression characteristics of GhMPK4 were analysed by qRT-PCR.The results showed that GhMPK4 was expressed in root,stem and cotyledon.In addition,F.oxysporum,salicylic acid(SA)and jasmonate(JA)inhibited the expression of GhMPK4 in different degrees.(4)The silencing of Gh MPK4 enchances the sensitivity to F.oxysporum in cotton.Using VIGS(Virus-induced gene silencing)technology,GhMPK4 silent cotton plants were obtained.Compared with the control group cotton,the silent cotton leaf accumulated more reactive oxygen species,the cell damage was more serious,and the SA-and JA-mediated expression of related genes was significantly down-regulated.(5)In tobacco plants,over-expression of GhMPK4 increases the sensitivity to F.oxysporum.Using agrobacterium-mediated leaf disc transformation method,three stable transgenic lines were selected as experimental materials.It was inoculated with F.oxysporum and found that compared with wild-type tobacco,overexpression plants accumulated more reactive oxygen species,cell damage was more serious,and the SA-and JA-mediated the expression of related genes was up-regulated.(6)Constitutive activation of GhMPK4 produces lesions phenotypes in cotton leaves.We have obtained different activities of GhMPK4(inactive GhMPK4 AA and constitutively active GhMPK4EE)by site directed mutagenesis.Further experiments revealed that over-activated GhMPK4 accumulated a large amount of reactive oxygen species,resulted in lesions,and enhanced the expression of plant resistance genes.(7)GhMPK4-GhWRKY33 cascade pathway plays the same role in regulating cotton resistance to Fusarium wilt.The downstream gene of GhMPK4,GhWRKY33 was screened by yeast two-hybrid.VIGS technology was used to obtain GhWRKY33 silent cotton plants.Through inoculation experiments,it was found that the silencing of GhWRKY33 enhanced the sensitivity to F.oxysporum in cotton.This phenomenon is consistent with the phenomenon that GhMPK4 silent cotton.